Our long term goal is to understand both the biogenesis and turnover mechanisms involved in the homeostasis of peroxisomes, an essential subcellular organelle that is intimately involved in many metabolic pathways, particularly lipid metabolism. Impairment of peroxisome biogenesis is the underlying cause of many fatal and debilitating human peroxisome biogenesis disorders (PBDs). Peroxisomal matrix proteins with the peroxisomal targeting signals, PTS1 or PTS2, are recognized by receptors, Pex5 and Pex7, respectively. The PTS2 pathway also uses co-receptors. In previous work, we and others demonstrated the dynamic behavior of some PTS receptors/co-receptors during the matrix protein import cycle, showing that they shuttle to and from peroxisomes. Receptor/co-receptor recycling and degradation are dependent on mono- and poly-ubiquitination steps, with the latter process also engaging the ubiquitin proteasome system (UPS). Peroxisomal membrane protein (PMP) biogenesis is poorly understood, with mPTSs serving as signals for peroxisomal membrane targeting. Whether PMPs are sorted to peroxisome membranes solely by their post- translation insertion into peroxisomes, or via the endoplasmic reticulum (ER), or possibly both, are ongoing debates. There is also considerable discussion regarding the relative contributions of the growth and division versus de novo biogenesis models for peroxisome biogenesis. Pex19 has been viewed as the mPTS receptor for the post-translational insertion of many PMPs into peroxisome membranes, but we found new roles for Pex3 and Pex19 in the budding of pre-peroxisomal vesicles (ppVs) from the ER. Other proteins, such as Pex3, Pex16L and Pex25 also play a role in de novo peroxisome biogenesis, division and inheritance. Aims1 and 2 of this proposal focus on the de novo biogenesis of peroxisomes from the ER investigating the roles of four proteins - Pex3, Pex16L, Pex19 and Pex25 in this process, including the assembly of two peroxisome membrane fission machineries that function in a spatiotemporally distinct manner to cause fission of tubular pre-peroxisomes at the ER and later to divide mature peroxisomes. Aim1 asks how Pex19 and Pex3 perform a role in budding of ppVs from the ER, focusing on Pex19 domains involved in ppV budding, testing specific hypotheses regarding Pex3 and Pex19 functions in budding of two classes of ppVs and in peroxisome inheritance, as well as the underlying mechanisms involved. Aim2 tests specific hypotheses regarding the roles of Pex16-like (Pex16L) and Pex25 proteins in either ppV budding and/or peroxisome division, with attention on the components of the fission machineries and their temporal action. Aim3 addresses PTS receptor dynamics, and its regulation by metabolic cues, focusing on Pex7, whose dynamics has not been studied to date. Aim3 also addresses whether Pex7 and cargo turnover are regulated by the UPS and metabolic cues. Answers to these questions will not only provide a mechanistic understanding of these processes, but also shed light on human disease states because all the proteins we focus on are conserved in humans and mutated in PBDs.